A Novel Electrochemiluminescence Immunosensor for the Analysis of

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A Novel Electrochemiluminescence Immunosensor for the Analysis of HIV-1 p24 Antigen Based on P-RGO@Au@Ru-SiO2 Composite Limin Zhou, Jianshe Huang, Bin Yu, Yang Liu, and Tianyan You ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b08154 • Publication Date (Web): 21 Oct 2015 Downloaded from http://pubs.acs.org on October 22, 2015

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ACS Applied Materials & Interfaces

A Novel Electrochemiluminescence Immunosensor for the Analysis of HIV-1 p24 Antigen Based on P-RGO@Au@Ru-SiO2 Composite Limin Zhoua,b，Jianshe Huanga, Bin Yua,c, Yang Liud, Tianyan Youa* a

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China b

c

University of the Chinese Academy of Sciences, Beijing, 100049, China

National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, 130012,China d

Nanochemistry Research Institute, Department of Chemistry, Curtin University, GPO Box U1987, Perth, Western Australia 6845, Australia

ACS Paragon Plus Environment

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ABSTRACT Ru(bpy)32+- doped silica (Ru-SiO2) nanoparticles and gold-nanoparticle-decorated graphene (PRGO@Au) were combined to form P-RGO@Au@Ru-SiO2 composite. And the composite was used to develop a novel sandwich-type electrochemiluminescence immunosensor for the analysis of HIV-1 p24 antigen. The composite worked as carrier to immobilize target antibody and to build a sandwich-type electrochemiluminescence immunosensor through the interaction between antigen and antibody. Importantly, high ECL signal could be obtained due to the large amounts of Ru(bpy)32+ molecules in per Ru-SiO2 nanoparticle. P-RGO@Au composite with good conductivity and high surface area not only accelerated the electron transfer rate, but also improved the loading of both ECL molecules and capture antibody, which could further increase the ECL response and result in high sensitivity. Taking advantage of both Ru-SiO2 nanoparticles and P-RGO@Au composite, the proposed immunosensor exhibited a linear range from 1.0 ×10-9 to 1.0 × 10-5 mg mL−1 with a detection limit of 1.0 ×10-9 mg mL−1 for HIV-1 p24 antigen. The proposed ECL immunosensor was used to analyze HIV-1 p24 antigen in human serum, and satisfactory recoveries were obtained, indicating that the proposed method is promising for practical application in clinical diagnosis of HIV infection.

KEYWORDS: Electrochemiluminescence; Immunosensor; HIV-1 p24 antigen; Graphene；Ru(bpy)32+- doped silica nanoparticle


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1. INTRODUCTION Acquired immune deficiency syndrome (AIDS) is a severe infectious disease caused by the human immune deficiency virus (HIV). Among the two types of HIV: HIV-1 and HIV-2, HIV-1 is the most common cause of AIDS.1 Since there are still no effective treatments, prevention of infection is the primary strategy to control the spread of HIV.2 Therefore, detecting HIV at an early stage will be great favorable for decreasing the probability of infection. Usually, analysis of HIV antibody in serum was used to the detection of HIV, but in the early stages of HIV infection when HIV antibody has not yet appeared in the sufferers’ serum (the window period), the patient is highly infectious.3 However, the HIV-1 capsid protein, the p24 antigen, appeared at an earlier stage of HIV infection than antibody, which is due to an explosive replication of the virus following acute infection and is correlated with highly infectious viraemia.2 Thus the p24 antigen can be used as an excellent biomarker in the diagnosis of HIV to efficiently shorten the “window period”. To date, many methods have been used to the analysis of p24, including enzyme-linked immunosorbent assay,4-5 electrochemical methods,6-9 fluoroimmunoassay10 and so on.11-14 However, it is still desirable and important to develop new and sensitive methods for the detection of p24 to predict AIDS diseases. As a powerful analytical method, electrochemiluminescence (ECL) method has been widely used in environmental monitoring,15-16 food safety17 and bioanalysis18-21 due to its high sensitivity, simple set-up and absence of background optical signal. Nowadays, with the development of nanotechnology, many nanomaterials have been used in ECL field to improve the analytical performance. Among them, SiO2 nanoparticles (NPs) have attracted much attention due to its special pore structure, high surface area and excellent biocompatibility. For example, Tan and co-workers prepared Ru(bpy)32+-doped silica (Ru-SiO2) nanoparticles and


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used as fluorescent probe for leukemia cell identification.22 Furthermore, the Ru-SiO2 nanoparticles were also used to construct ECL sensor for DNA analysis,23-24 protein detection,25 immunoassay,26-27 cell test28-29and so on.30 Excellent analytical performances were obtained since each Ru-SiO2 nanoparticle contained large amounts of Ru(bpy)32+ molecules which could greatly enhance the ECL signal and result in high sensitivity. However, the low conductivity of Ru-SiO2 nanoparticle limits its high loading to some extent. On the other hand, graphene, a new class of two-dimensional (2D) carbon nanostructure, has attracted much attention in recent years due to its unique properties, such as high mechanical strength, high surface area, and rapid electron transfer. It has been widely used in the fields of photoelectric devices, fuel cells and so on.31 Recently, especially for its fantastic electrical conductivity, graphene was selected as an ideal material to construct ECL sensors.32-37 However, there has been no report on using both Ru-SiO2 nanoparticle and P-RGO@Au composite for ECL system. Herein, we firstly combine Ru-SiO2 nanoparticles and P-RGO@Au composite to prepare PRGO@Au@Ru-SiO2 composite. The composite was used as ECL label to build a novel electrochemiluminescence immunosensor for HIV-1 p24 analysis. The as-prepared nano composite takes advantage of both high loading of Ru(bpy)32+ in Ru-SiO2 nanoparticles and excellent conductivity and high surface area of P-RGO@Au composite, which could greatly amplify the ECL signal and improve the analytical sensitivity. To our knowledge, this is the first time to use the ECL method for the analysis of p24. The proposed immunosensor exhibited wide linear range and low detection limit for HIV-1 p24 antigen with good stability and reproducibility. Good recoveries were obtained when the immunosensor was used to the analysis of HIV-1 p24 antigen in human serum. The proposed method provides a promising alternative for HIV-1 p24 antigen analysis in real samples.


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2. MATERIALS AND METHODS 2.1. Materials and Reagents Triton X-100 (TX-100), Bovine Serum Albumin (BSA) and Alpha fetoprotein (AFP) were supplied by Beijing Dingguo Biotechnology Co.Ltd (Beijng, China). Tetraethyl orthosilicate (TEOS) ， Tris

(2,2-bipyridyl)

dichlororuthenium(II)

hexahydrate

(Ru(bpy)3Cl2.6H2O),

Poly(diallyldimethylammonium chloride) (PDDA), tripropylamine (TPA)，HAuCl4 and sodium citrate were purchased from Aldrich. Ammonium hydroxide (25-28 wt%), cyclohexane and nhexane were bought from Beijing Chemical Reagent Factory (Beijing, China). 1-hexanol and sodium borohydride were offered by Sinopharm Chemical Reagent Co.Ltd (Shanghai, China). Carcinoembryonic antigen (CEA), CEA primary antibody (CEA-Ab1), CEA secondary antibody (CEA-Ab2), p24 primary antibody (p24-Ab1), p24 secondary antibody (p24-Ab2) and p24 antigen were provided by Linc-Bio Science Co. Ltd (Shanghai, China). Human serum was provided by healthy volunteer. Phosphate buffer solutions (PBS) were prepared with Na2HPO4 and NaH2PO4. The washing buffer was 0.05% (w/v) Tween-20 in 0.01 M pH 7.4 PBS. All aqueous solutions in the experiments were prepared with doubly distilled water. 2.2. Apparatus The ECL responses were measured on a MPI-E ECL analyzer (Xi'An Remax Electronic Science & Technology Co. Ltd., China) with a voltage of 800 V supplied to the photomultipliertube (PMT). CV behaviors were recorded using a CHI832 voltammetric analyzer (Shanghai Chenhua Apparatus Inc., China). An Ag/AgCl (saturated KCl) electrode and a platinum wire worked as reference electrode and counter electrode, respectively. Modified glassy carbon electrode was used as working electrode. UV-visible spectra were measured on UV mini 1240 (Shimadzu Instruments, Japan). Transmission electron microscopy (TEM) was conducted


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using the Hitachi H-600 transmission electron microscope (TEM) operated at 100 kV. AUTOLAB PGSTAT302N was used to record the electrochemical impedance Spectroscopy (EIS). 2.3. Preparation of P-RGO@Au@Ru-SiO2 and Secondary Antibody Bioconjugate To obtain the ECL bioconjugate, firstly, Ru-SiO2 and P-RGO@Au were prepared respectively. Then, Ru-SiO2 and P-RGO@Au were combined to prepare P-RGO@Au@Ru-SiO2 composite. At last, the composite interacted with secondary antibody to form the bioconjugate. Ru-SiO2 nanoparticles were prepared by the microemulsion method according to previous report.38 Firstly, 5.31 mL of TX-100, 22.5 mL of cyclohexane, 5.4 mL of 1-hexanol and 1.02 mL of water were mixed together to form the water-in-oil microemulsion. And then 240 µL of 0.1 M Ru(bpy)32+ aqueous solution and 300 µL of TEOS was added successively. A polymerization reaction was initiated by adding 180 µL of NH4OH. After 24 h, the Ru-SiO2 nanoparticles were isolated by acetone. After centrifuging and washing with ethanol and water for several times to remove surfactant molecules, the yellow Ru-SiO2 nanoparticles were obtained. Graphite oxide (GO) used in the experiments was synthesized using the modified Hummers method.39 P-RGO was obtained by the reduction of GO according to previous literature 40 with a little change. Briefly, 50 mg of GO was added to 50 mL of 0.5 wt% PDDA solution and sonicated until it became clear. Subsequently, 5 µL of 85% hydrazine hydrate was added to the solution and it was heated at 95 °C for 24 h. At last, a homogeneous black suspension was obtained. The final product was collected and dried at 60 °C for 24 h and it was donated as PRGO. The P-RGO@Au composite was synthesized through the NaBH4 reduction method according to a previous report41 with a little change. Firstly, 1 mg P-RGO was dispersed in 5 mL of 1 mM


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HAuCl4 solution by sonication. Then 5 mL of 40 mM NaBH4 solution was added dropwise to the mixture solution under stirring. After continuously stirring for 30 min, the resulting P-RGO@Au composites were collected by centrifugation and washed with deionized water. At last, the composites were dispersed in 4 mL water. To obtain the P-RGO@Au@Ru-SiO2 composite, Ru-SiO2 nanoparticles were added to 5 ml PRGO@Au composite solution with mild ultrasonication and the final concentration of Ru-SiO2 nanoparticles was 0.1 mg mL-1. After stirring for 24 hours, the mixture was centrifuged and washed with water for three times. The P-RGO@Au@Ru-SiO2 composite was dispersed in 1 mL water lastly. The p24 secondary antibody (p24-Ab2) conjugated P-RGO@Au@Ru-SiO2 nanocomposite (p24-Ab2 bioconjugate) were prepared by adding 50 µL of 150 µg mL-1 p24-Ab2 to the P-RGO@Au@Ru-SiO2 solution. After incubating at 4 °C for 24 hours, the product was centrifuged and washed with PBS. At last, the bioconjugate were dispersed in 0.5% BSA. The PRGO@Au@Ru-SiO2@CEA-Ab2 (CEA-Ab2 bioconjugate) was obtained with the same procedure. 2.4. Fabrication of the ECL Immunosensor The fabrication process of the immunosensor was shown in Figure 1. Firstly, GCE (3 mm) was polished with 0.3 µm and 0.05 µm alumina powders, respectively. Then it was sonicated in ethanol and water thoroughly. After drying at room temperature, Au NPs modified GCE (Au/GCE) was prepared by dropping 5 µL Au NPs (the preparation of Au NPs was shown in supplementary materials) onto the GCE. Subsequently, the Au/GCE electrode was immersed in 80 µL of 50 µg mL-1 p24-Ab1 at 4 °C for 24 h to immobilize p24-Ab1 (p24-Ab1/Au/GCE). After washing with buffer to remove the physically adsorbed Ab1, the modified electrode was incubated with 80 µL of 1% BSA at 37 °C for 1 h to block the nonspecific binding sites


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(BSA/p24-Ab1/Au/GCE). Followed by washing again, the electrode was immersed in 80 µL of p24 at 37 °C for 2.5 h to capture the p24 antigen (p24/BSA/p24-Ab1/Au/GCE). Finally, the sandwich immunosensor was fabricated completely by incubating the electrode with 80 µL of p24-Ab2 bioconjugate at 37 °C for 2.5 h (p24-Ab2 bioconjugate/p24/BSA/p24-Ab1/Au/GCE). The obtained immunosensor was investigated in 0.1 M PBS (pH 7.5) containing of 5 mM TPA as coreactant. And it was stored at 4 °C when not in use. The immunosensor for CEA antigen was constructed in the same way using corresponding CEA antibodies.

Figure 1. (A) The preparation process of p24-Ab2 bioconjugate and (B) the fabrication process of the proposed immunosensor.

3. RESULTS AND DISCUSSION 3.1. Characteristics of Different Nanomaterials


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The Ru-SiO2 nanoparticles were synthesized using a water-in-oil reverse micromulsion method. Many Ru(bpy)32+ molecules were encapsulated while the SiO2 nanoparticles were formed. The obtained nanoparticles were spherical and uniform in size with 55±3 nm in diameter (Figure 2A). As for P-RGO@Au composite, the Au NPs were homogeneously dispersed on the graphene sheet (Figure 2B). Since Ru-SiO2 nanoparticles were negative charged while P-RGO@Au composite was positive charged, the P-RGO@Au@Ru-SiO2 composite was successfully produced through electrostatic interaction when combining Ru-SiO2 nanoparticles with PRGO@Au composite (Figure 2C). (The zeta potential of Ru-SiO2 nanoparticles, P-RGO@Au composite and P-RGO@Au@Ru-SiO2 composite were shown in supporting information).

Figure 2. TEM image of (A) Ru-SiO2 nanoparticles, (B) P-RGO@Au composite and (C) PRGO@Au@Ru-SiO2 composite; (D) UV-vis absorption spectra of Ru-SiO2 nanoparticles, PRGO@Au composite and P-RGO@Au@Ru-SiO2 composite.


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The UV-vis absorption spectra of Ru-SiO2 nanoparticles, P-RGO@Au and P-RGO@Au@RuSiO2 composite were investigated (Figure 2D). There were two obvious peaks at about 287 nm and 457 nm for Ru-SiO2 nanoparticles, which were assigned to ligand-centered transitions and metal-to-ligand charge transfer (MLCT) of Ru(bpy)32+ molecule, respectively.42 For P-RGO@Au composite, the peak at 260 nm can be associated with to π–π* transitions of aromatic C–C bonds,41 and the characteristic peak of Au NPs at 530 nm indicated that the Au NPs was successfully assembled on the graphene sheet. After combining the P-RGO@Au composite with Ru-SiO2 nanoparticles, the characteristic peaks of both Ru(bpy)32+ and Au NPs were observed, indicating that P-RGO@Au@Ru-SiO2 composite was successfully prepared. 3.2. Feasibility of the Designed Immunosensor Using CEA as Target Carcinoembryonic antigen (CEA) is one of the most common cancer biomarkers. Thus, we firstly used CEA as model antigen to evaluate the feasibility of the designed immunosensor. As shown in Figure S1, a low ECL signal was produced without target CEA antigen (curve a). However, in the presence of 100 ng mL-1 CEA, ECL intensity was greatly enhanced (curve b), which was due to that more ECL probe was immobilized on the electrode surface through the interaction between antigen and antibody. This result indicated that the ECL response of the immunosensor was related to the target antigen. Furthermore, the ECL intensity of the immunosensor was negligibly changed over continuous 15 cyclic potential scans, suggesting the good stability of the immunosensor. These data above showed that the immunosensor could be used to the analysis of target antigen. 3.3. Characterization of the Immunosensor for the Analysis of p24 Antigen


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HIV-1 p24 antigen, which appears at an earlier stage of HIV infection than antibody, is an excellent biomarker for the diagnosis of HIV. As the analysis of p24 antigen can greatly decrease the probability of HIV infection. Therefore, the proposed immunosensor was applied to the analysis of p24 antigen. Firstly, the fabrication process of the immunosensor was investigated. The cyclic voltammetry response of each step in 0.1 M KCl solution containing of 5.0 mM [Fe(CN)6]3-/4- was shown in Figure 3A. A couple of redox peaks of [Fe(CN)6]3-/4- were observed on bare GCE electrode (curve a). The peak currents increased after coating Au NPs on the electrode (curve b) due to the larger electrochemically active area.43 When Au/GCE was incubated with p24-Ab1, the peak currents decreased (curve c), since the insulated p24-Ab1 was assembled on the electrode via the interaction between the Au NPs and the amino groups of p24-Ab1, which hindered the electron transfer process. The SEM images in Figure S2 showed that the electrode was covered with a layer of dense protein when incubated with p24-Ab1, which indicated that p24-Ab1 was successfully assembled on the electrode surface. Additionally, subsequent surface blocking with BSA (BSA/p24-Ab1/Au/GCE, curve d) and incubating with p24 (p24/BSA/p24-Ab1/Au/GCE, curve e) lead to further decreased peak currents and broadened peak-to-peak potential difference (∆Ep), which can be attributed to the increased electron resistance caused by those insulated biomolecules. Finally, when the modified electrode was incubated with p24-Ab2 bioconjugate (p24-Ab2 bioconjugate/p24/BSA/p24-Ab1/Au/GCE, curve f), further decrease of peak currents and the increase of ∆Ep were observed. Although the P-RGO@Au composite has good conductivity, both the Ru-SiO2 nanoparticles and protein molecules in the bioconjugate were nonconductive, which resulted in sluggish electron transfer kinetics and low electrochemical responses.


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Figure 3. (A) CV curves and (B) Corresponding EIS responses of (a) GCE, (b) Au/GCE, (c) p24-Ab1/Au/GCE, (d) BSA/p24-Ab1/Au/GCE, (e) p24/BSA/p24-Ab1/Au/GCE, (f) p24-Ab2 bioconjugate/p24/BSA/p24-Ab1/Au/GCE in 0.1 M KCl solution containing of 5.0 mM [Fe(CN)6]3-/4-.

Electrochemical impedance spectroscopy (EIS) was performed at each step in 0.1 M KCl solution containing of 5.0 mM [Fe(CN)6]3-/4- to understand the fabrication process of the proposed sensor. As shown in Figure 3B, when coated with Au NPs (Au/GCE, curve b), the electrode showed decreased electron transfer resistance (Rct) as compared with bare GCE (curve a), since Au NPs with good conductivity could improve the electron transfer rate. However, the


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Rct increased obviously after successive assembly of p24-Ab1, BSA, p24 and p24-Ab2 bioconjugate onto the electrode, which was attributed to the blocking effect of insulated protein molecules on the electron transfer process. These Rct changes of each step in EIS responses were consistent with the CV results. The ECL response of different nanocomposite used as ECL probe was shown in Figure 4. When the Ru-SiO2 nanoparticles directly interacted with p24-Ab2 to build the immunosensor, a low ECL signal was produced (curve a), which was related to the insulated SiO2 nanoparticles that hindered the electron transfer to some extent. In addition, there was no special interaction between the Ru-SiO2 nanoparticles and p24-Ab2, thus little Ru-SiO2 nanoparticles were immobilized on the electrode, resulting in low ECL response. In contrast, when P-RGO@RuSiO2 composite was used as ECL probe, the ECL intensity greatly increased (curve b). Because the addition of P-RGO not only worked as carrier for loading Ru-SiO2 nanoparticles and antibody, but also improved the conductivity and accelerated the electron transfer rate. In addition, the ECL signal further increased when the P-RGO@Au@Ru-SiO2 composite was used (curve c). This was because that the Au NPs could interact with p24-Ab2,44 which enabled p24Ab2 to firmly assemble p24-Ab2 on the composite with increased absorption amount. Thus, more ECL probe could be immobilized on the electrode surface through the interaction between antigen and antibody to enhance the ECL signal. These results indicated that P-RGO@Au@RuSiO2 composite could be used as an excellent probe to construct ECL immnosensor.


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Figure 4. ECL−time profiles of p24/BSA/p24-Ab1/Au/GCE with (a) Ru-SiO2, (b) P-RGO@RuSiO2, (c) P-RGO@Au@Ru-SiO2 as ECL probe incubating with 1×10-4 mg mL-1 p24. Electrolyte: 0.1M PBS (pH 7.5) containing of 5 mM TPA; Scan rate: 100 mV s-1. Scan potential: 0~1.35V. The voltage of the photomultiplier tube was set at 800 V.

3.4. Optimization of Experimental Parameters Firstly, the effect of concentration of Ru-SiO2 for preparing P-RGO@Au@Ru-SiO2 composite on the ECL signal was investigated. Certain amount of P-RGO@Au composite and different concentrations of Ru-SiO2 were mixed to prepare P-RGO@Au@Ru-SiO2 composite. As shown in Figure S3A, ECL signal increased with the higher Ru-SiO2 concentration, as more luminophors were assembled on the composite. However, when the Ru-SiO2 concentration was more than 0.1 mg mL-1, no obvious signal enhancement was produced, indicating that Ru-SiO2 was saturated on the composite. Therefore, 0.1 mg mL-1 Ru-SiO2 was used to prepare PRGO@Au@Ru-SiO2 composite. The effect of the dosage of p24-Ab2 (150 µg mL-1) on the ECL responses was also evaluated, and the corresponding ECL responses were shown in Figure S3B. With increasing volume of


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p24-Ab2 from 10 µL to 50 µL, the ECL intensity increased and then reached a plateau. Therefore, 50 µL was used for the preparation of bioconjugate. Since Ru(bpy)32+-TPA system is a pH-dependent reaction, we investigated the effect of pH on ECL intensity (Figure S3C). The ECL intensity increased accordingly with the increase of pH value from 6.5 to 7.5. The ECL response began to fall thereafter. This was because too high pH value resulted in the decomposition of some species, leading to a diminished ECL reagent available for ECL reaction. 38 Therefore, pH 7.5 was selected as optimal pH value. The ECL intensity of the immunosensor was also affected by the incubation time of antibody and antigen. As indicated in Figure S3D, the ECL intensity enhanced with longer incubation time and reached its maximum at 150 min. There was no obvious signal increase with longer incubation time, which indicated the saturation of antigen on the immunosensor surface.45 Therefore, 150 min was chosen as the optimal incubation time. 3.5. Analysis of p24 Antigen Under the optimal condition, the proposed immunosensor was used to the analysis of p24 antigen. The analytical performance was investigated by incubating the immunosensor in different concentration of p24. As exhibited in Figure 5A, the ECL signal enhanced accordingly with the increase of the p24 concentrations. The ECL intensity (I) was proportional to the logarithmic value of the p24 concentration ranging from 1.0 ×10-9 to 1.0 × 10-5 mg mL−1. The linear regression equation is I = 185.3 × log Cp24 + 2126.1 with a correlation coefficient of 0.997. And the detection limit was 1.0 ×10-9 mg mL−1. Table 1 compares the analytical performance of proposed immunosensor with other methods. Although the lowest LOD was obtained with the plasmonic ELISA assay method 4, the immunosensor proposed in this work achieved wider linear range and lower LOD as compared with amperometric immunosensor


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based on GNP/CNT/AEP modified electrode 1, direct gold electroplating-modified electrode 2, polyelectrolyte/gold magnetic nanoparticle assembly modified electrode

48

and DMA-NAS-

MAPS composite enhanced fluorescence sensor 10, our strategy is promising for the development of effective immunosensors with high performances. The excellent analytical performances of the proposed method were ascribed to the signal amplification of the P-RGO@Au@Ru-SiO2 composite. Each Ru-SiO2 nanoparticle contained large amounts of Ru(bpy)32+ molecules which could produce high ECL intensity. The PRGO@Au composite with good conductivity and high surface area not only improved the electron transfer rate of the system but also increased the loading of both ECL molecules and capture antibody, which could increase the ECL response further. The P-RGO@Au@Ru-SiO2 composite as ECL probe combined these advantages and produced high ECL signal, thus resulting in good analytical performance for analysis of p24 antigen.


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Figure 5. (A) Calibration curve of ECL intensity to logarithmic p24 antigen concentration. Insert: ECL− time curves of the immunosensor incubating with 1.0 ×10-9 mg mL−1, 1.0 ×10-8 mg mL−1, 5.0 ×10-8 mg mL−1, 1.0 ×10-7 mg mL−1, 1.0 ×10-6 mg mL−1, 5.0 ×10-6 mg mL−1, 1.0 ×10-5 mg mL−1 p24 antigen. (B) Selectivity and specificity of the proposed immunosensor: BSA (2.0 ×10-5 mg mL−1), CEA (2.0 ×10-5 mg mL-1), AFP (2.0 ×10-5 mg mL-1), serum and p24 antigen (1.0 ×105

mg mL-1). I was the ECL intensity in the presence of the target or interferences and I0

represented the ECL signal of the blank. The detection conditions are the same as in Figure.4.

To investigate selectivity and specificity, the immunosensor was incubated with 2.0 × 10-5 mg mL−1 BSA, AFP, CEA and human serum, respectively. As displayed in Figure 5B, the ECL response of BSA, AFP, CEA and serum were negligible as compared with that of p24 antigen, which showed that the proposed immunosensor had good selectivity and specificity. What’s more, the relative standard deviation (RSD) of five immunosensors, which were fabricated by the same batch of probe, was 3.7%. And the RSD of three immunosensors, which were fabricated by the different batches, was 5.2%. These data indicated good fabrication reproducibility of the immunosensor. The inter-day and intra-day RSD of an immunosensors were 3.4% and 3.1%. In addition, the long-time stability of the immunosensor was tested by measuring the ECL response every 3-4 days and the immunosensor was kept at 4 °C when not in use. The ECL intensity still maintained 78% of the original response after one month, which exhibited acceptable long-time stability. 3.6. Analysis of p24 in Human Serum To evaluate the applicability in real sample, the proposed immunosensor was used to detect p24 antigen in three human serum samples. Human serum was diluted 100 times with PBS buffer and p24 antigen was added with a final concentration of 1.0 × 10-6 mg mL−1 and 1.0 × 10-8 mg


17


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mL−1 respectively. The recoveries were between 96.3 % and 106.2 % (Table 2), indicating that the developed immunosensor had the potential to detect p24 antigen in real samples.

Table 1 Comparison of analytical performance of proposed immunosensor with other methods. Method

Linear range (mg mL-1)

Detection limit (mg mL-1)

Electrochemical immunosensor based on Fe3O4@SiO2 1.0×10-9 -1.0×10-5 nanomagnetic probes and nanogold colloid-labeled enzyme–antibody copolymer as signal tag

Reference

5×10-10

(46)

1.0×10-10

(47)

Plasmonic ELISA assay with the naked eye

1.0×10-15

(4)

Amperometric immunosensor 1.0×10-8 -1.0×10-4 based on a direct gold electroplating-modified electrode

8.0×10-9

(2)

Amperometric immunosensor 1.0×10-8 -6.0×10-5 based on GNP/CNT/AEP modified electrode

6.4×10-9

(1)

DMA-NAS-MAPS composite enhanced fluorescence signal

5.4×10-8

(10)

Amperometric immunosensor 1.0×10-7-1.0×10-4 based on a polyelectrolyte/gold magnetic nanoparticle assembly modified electrode

5.0×10-8

(48)

ECL immunosensor based on P- 1.0×10-9 -1.0×10-5 RGO@Au@Ru-SiO2 composite as signal label

1.0×10-9

This work

Nanoparticle-based amplification assay

bio-barcode 1.0×10-10 -1.0×10-6


18

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Table 2 Recovery results of the proposed immunosensor in human serum. Samples

Added (10-8 mg mL-1)

Found (10-8 mg mL-1)a

Recovery (%)

Serum 1

1.00

0.993

99.3

Serum 2

1.00

1.006

100.6

Serum 3

1.00

1.017

101.7

Serum 1

100

96.28

96.3

Serum 2

100

102.0

102.0

Serum 3

100

106.2

106.2

a

Average value from six successive measurements.

4. CONCLUSION In summary, a novel sandwich-type electrochemiluminescence immunosensor was developed using P-RGO@Au@Ru-SiO2 composite as ECL label. The immunosensor was used for the analysis of HIV-1 p24 antigen. Since P-RGO@Au@Ru-SiO2 composite takes advantages of high Ru(bpy)32+ loading property of Ru-SiO2 nanoparticles and the good conductivity and high surface area of P-RGO@Au, ECL signal was greatly amplified and resulted in excellent analytical performance. Wide linear range and low detection limit were obtained. What’s more, when the immunosensor was used to the analysis of HIV-1 p24 antigen in human serum, good recoveries were obtained. The proposed method provides a promising alternative for the determination of HIV-1 p24 antigen in real samples.

ASSOCIATED CONTENT Supporting Information.


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Page 20 of 27

Preparation of Au NPs, ECL response of the immunosensor in the absence and presence of CEA antigen, optimization of experimental parameter, SEM images of ITO, Au/ITO, Ab1/Au/ITO and BSA/Ab1/Au/ITO. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail address: [email protected]. Tel & Fax: +86-431-85262850.

ACKNOWLEDGMENT This work was supported by National Science Foundation of China (21222505, 21475124) and China Postdoctoral Science Foundation (2014T70301). REFERENCES (1).Kheiri, F.; Sabzi, R. E.; Jannatdoust, E.; Shojaeefar, E.; Sedghi, H. A Novel Amperometric Immunosensor Based on Acetone-extracted Propolis for the Detection of the HIV-1 p24 Antigen. Biosens. Bioelectron. 2011, 26, 4457-4463. (2).Zheng, L.; Jia, L. Y.; Li, B.; Situ, B.; Liu, Q. L.; Wang, Q.; Gan, N. A Sandwich HIV p24 Amperometric Immunosensor Based on a Direct Gold Electroplating-Modified Electrode. Molecules 2012, 17, 5988-6000. (3).Gurtler, L.; Muhlbacher, A.; Michl, U.; Hofmann, H.; Paggi, G. G.; Bossi, V.; Thorstensson, R.; Villaescusa, R. G.; Eiras, A.; Hernandez, J. M.; Melchior, W.; Donie, F.; Weber, B. Reduction of the Diagnostic Window with a New Combined p24 Antigen and Human Immunodeficiency Virus Antibody Screening Assay. J. Virol. Methods 1998, 75, 27-38.


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TOC


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A Novel Electrochemiluminescence Immunosensor for the Analysis of

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